L-serine is an amino acid that currently is used in the cosmetics, pharmaceutical and medical industry. The estimated annual production of serine is between 300-1000 tons (Leuchtenberger et al., 2005). The compound has also been identified as one of the top 30 most interesting biochemicals because of its potential use as a building block biochemical. The current production is based on conversion of glycine and methanol using resting cells (Hagishita et al., 1996), where methylotrophs convert methanol to fomaldehyde and transfer the CH2—OH unit of the molecule to glycine using serine hydroxymethyltransferase (glyA). This fermentation process is time consuming and glycine is an expensive starting material. Developing a method for producing serine at low cost directly from glucose is therefore attractive.
Serine has the potential to be made from glucose by fermentation with a very high theoretical yield (Burgard and Maranas, 2001). However, several challenges need to be addressed in order to increase the yield, the most crucial one being degradation of serine in the production organism.
Serine has two key degradation pathways in E. coli. Serine to pyruvate catabolism is in E. coli catalyzed by three deaminases namely sdaA, sdaB and tdcG, while C. glutamicum only has one deaminase (sdaA) with activity towards serine. In both organisms, the conversion of serine to glycine is encoded by glyA. Serine production by knocking out only deaminases has been attempted in E. coli (U et al., 2012) and C. glutamicum (Peters-Wendisch et al., 2005). In E. coli transient accumulation of 3.8 mg/L from 1 g/L glucose was observed when only one of the pathway gene (serA) was overexpressed. Deletion of the deaminase on C. glutamicum lead to marginal and transient increase in the serine titer. In recent studies, E. coli was engineered to enhance the flux of 3-phosphoglycerate by perturbing the TCA-cycle and glyoxylate shunt (Gu et al., 2014). The resulting strain, where only one deaminase, was removed (sdaA) was reported to produce 8.45 g/L serine from 75 g/L glucose (11.2% yield).
Down regulation of glyA (Peters-Wendisch et al., 2005) in C. glutamicum resulted in the production of 9 g/L serine from 40 g/L glucose but lead to an unstable strain. glyA is an important enzyme that converts serine to glycine and in this step transfers one carbon unit to tetra hydrofolate (THF), which is used as cofactor. Removal of the folic acid pathway and supplementation of folic acid lead to a stable C. glutamicum, and a production of 36 g/L serine, however with a relatively low yield (Stolz et al, 2007).
Deletion of both of the major serine degradation pathways (serine to pyruvate and serine to glycine) has not been previously been achieved. It is furthermore known that serine becomes toxic even at low concentrations in strains that lack the pyruvate degradation pathway (Zhang and Newman, 2008). It is expected that serine may inhibit the production of branched amino acids in E. coli Hama et al., 1990), and the conversion of serine to hydroxypyruvate and acrylates, which is toxic to the cell (de Lorenzo, 2014). For efficient production of L-serine or a derivative thereof, it is therefore desirable to both remove the serine degradation pathways and address the problems associated with toxicity of serine.